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VOL. 51, 1964 BIOCHEMISTRY: SCfMITT ET AL. 493
(^2) Epstein, R. H., et al., in Synthesis and Structure of Macromolecules, Cold Spring Harbor Sym- posia on Quantitative Biology, vol. 28 (1963), in press. (^3) Garen, A., personal communication. 4Gorini, L., and H. Kaufman, Science, 131, 604 (1960). 6 Davis, B. D., and E. S. Mingioli, J. Bacteriol., 60, 17 (1950). (^6) Gorini, L., Bull. Soc. Chim. Biol. (France), 40, 1939 (1958). 7Wollman, E. L., and F. Jacob, Ann. Inst. Pasteur, 95, 641 (1958). (^8) Beckwith, J. R., Biochim. Biophys. Acta, 76, 162 (1963); and contribution to Genetics Con- ference in Gatersleben (E. Germany), August 1963, in press. (^9) Pardee, A. B., J. Bacteriol., 73, 376 (1957). (^10) We are indebted to Dr. L. Cavalli for this information. (^11) We are indebted to Dr. M. Meselson for this information. (^12) Campbell, A., Virology, 14, 22 (1961). (^13) Spotts, C. R., and R. Y. Stanier, Nature, 192, 633 (1961). (^14) Spotts, C. (^) R., J. Gen. Microbiol., 28, 347 (1962). 16 Flaks, J. B., et al., Biochem. Biophys. Res. Comm., 7, 340 (1962). (^16) Davies, J.^ E., unpublished results.
THE ANTIGENICITY OF TROPOCOLLAGEN*
BY F. 0. SCHMITT, L. LEVINE,t M. P. DRAKE,^ A. L.^ RUBIN,$ D.^ PFAHL, AND P. F. DAVISON DEPARTMENT OF BIOLOGY MASSACHUSETTS INSTITUTE OF^ TECHNOLOGY, DEPARTMENT OF BIOCHEMISTRY, BRANDEIS UNIVERSITY, AND THE^ ROGOSIN LABORATORIES, DEPARTMENT OF MEDICINE, CORNELL UNIVERSITY MEDICAL^ CENTER Communicated January 28, 1964 Collagen is a structural protein which constitutes about 30 per cent by weight of all protein in the mammalian body. The^ collagen fiber^ itself^ is^ relatively insolu- ble, but various conditions of solvent, pH, ionic environment,' and temperature may be used to obtain soluble fractions. Physical characterization of these soluble fractions, and X-ray and electron micrograph studies on precipitates obtained under specific conditions, have shown that the native collagen fiber is built up by the highly ordered aggregation of a fundamental unit termed the tropocollagen (TC) molecule,3-5 which is a stiff rod with a length of approximately 2800 A and a
molecular weight of about 300,000. The -700 A^ periodicity seen in^ electron
micrographs of collagen fibrils is believed to reflect the aggregation of the tropocol- lagen monomers in a polarized quarter-stagger array. The internal structure of the TC molecule is characterized by three polypeptide chains wound in a triple helix.7 The basic structural unit is the a-chain-a poly- peptide strand with a molecular weight of approximately 100,000.8 Two a-chains
may be cross-linked to form a ,8-chain, and three to form a y-chain.9' 10
The processes initiating and controlling the formation of a^ native-type fibril in vivo from TC monomers are unknown.^ It^ might proceed via^ the lateral^ aggrega- tion of staggered monomers^ with^ a^ consequent progressive build-up^ of^ a^ filament, or it could proceed via the lateral aggregation of protofibrils which form initially by end-to-end polymerization of the monomers. It was demonstrated in this laboratory that sonic irradiation modifies the normal^ end-to-end^ interactions of
the monomers, and^ it was^ proposed that TC^ molecules^ bear^ peptide "end-chains"
494 BIOCHEMISTRY:^ SCHMITT^ ET^ AL.^ PROC.^ N. A.^ S.
which by^ specific^ interaction^ mediate^ the^ linear^ polymerization^ of^ monomers^ into protofibrils. I Hodge and^ Petruskal2^ have^ recently^ shown^ that^ successive^ TC^ monomers^ in^ the
collagen fibril overlap^ instead^ of^ aggregating^ end-to-end.^ It^ would^ appear,^ there-
fore, that if^ peptide^ appendages^ on^ the^ body^ of^ the^ collagen^ molecule^ underlie specificity of^ polymerization,^ these^ structures^ may^ not^ reside^ exclusively^ at^ the end of the molecules,^ and^ the^ term^ "end-chains"^ with^ its^ terminal^ connotation^ has been replaced by^ the term^ "telopeptides."'" Studies in^ this laboratory have^ shown^ that^ small^ peptide^ fragments^ can^ be^ split
from TC by^ proteases^ and^ with^ their^ removal^ the^ aggregative^ properties^ of^ the^ TC
molecules are modified^ without^ substantial^ alteration^ of the^ triple-helix^ body^ of^ the highly elongate TC^ macromolecule."3-6^ On^ the basis^ of^ these^ observations^ it^ was suggested that homeostatic^ control^ of TC^ interaction^ and^ hence^ of^ deposition^ and degradation of collagen fibrils^ may^ be^ achieved^ in^ the^ organism^ by^ enzymatic
modification of telopeptides.'3^ The^ telopeptides^ contain^ a^ large^ fraction of^ all^ the
tyrosine in TC; their amino^ acid^ composition differs^ distinctly^ from^ that of^ bulk collagen.
It is well known that^ collagen^ is^ at^ best^ a^ weak^ antigen.^ In view of^ the^ similarity
of the X-ray diagrams obtained^ from^ collagens^ isolated^ from^ a^ variety^ of^ animal species, it would appear that^ the^ structure^ of^ the^ molecule^ is^ very^ similar^ in all species examined thus far.^ With this^ in^ mind^ and^ in^ view of the^ high^ tyrosine
content of the^ telopeptides^ we^ were^ led^ to^ postulate^ that^ most^ if^ not^ all the^ antigen-
icity of^ collagen^ might^ be^ ascribable^ to^ the^ telopeptides.'3^ The^ experiments^ de- scribed below^ substantiate^ this^ postulate. Experimental.-Physical and^ chemical^ methods^ of^ analysis^ have been^ described
previously.'13, 1
Collagen preparation:^ Calf-skin^ collagen^ was^ prepared^ by^ a^ procedure^ detailed elsewhere."5 In^ essence^ the^ procedure^ consisted^ of^ reprecipitating^ citrate-extracted, 1 per cent sodium^ chloride-insoluble^ TC^ until^ the^ preparation^ was^ rendered^ pure as judged by the^ criteria^ of^ amino acid^ analyses^ and^ electrophoretic^ homogeneity. Protease treatment:^ The TC^ was^ digested^ with^ pepsin^ (1:^100 by^ weight,^ or^ 1:^8 mole/mole of^ enzyme:^ substrate)^ in^ 0.05 per^ cent^ acetic^ acid^ (pH^ 3.5)^ at^200 for
24-96 hr.^ Peptide^ fragments^ were^ removed^ by^ dialysis,^ and^ the^ TC^ was^ separated
from the pepsin^ by^ free^ diffusion^ electrophoresis'3^16 or^ by^ precipitation^ of the^ TC
by 15 per cent^ KCl.'8^ Pronase^ treatment^ was^ performed^ at^ pH^ 7.2^ in^ 0.1^ molar
calcium acetate (at^ a^ similar^ enzyme:^ substrate^ ratio)^ at^200 for^ 24-96^ hr.^ The
TC was^ recovered^ by KCl^ precipitation^ as^ mentioned. Antibody production: Antibodies^ to^ calf-skin^ collagen^ were^ prepared^ by^ inject-
ing 2 ml^ of 0.35^ per^ cent TC^ solution^ in^ complete^ Freund's^ adjuvant^ intramuscularly
and into the toe pads^ of^ three^ rabbits.^ The^ rabbits^ were^ bled^ three^ weeks^ after
injection. The^ antibody^ concentration^ was^ measured^ by^ complement^ fixation.
Results.-All three^ rabbits^ produced^ antibodies^ to^ the^ injected^ TC.^ The^ comple- ment-fixation curve^ of^ the^ reaction^ between^ TC^ and^ one^ antiserum^ is^ shown^ in Figure 1. After pepsin digestion approximately^ 1 per^ cent^ of the^ TC molecule^ becomes
dialyzable, and some^ of the^ ,-^ and^ y-cross-links^ are^ removed.^ An^ almost^ insig-
nificant loss of optical^ rotation^ accompanies^ this^ change,^ and^ the^ intrinsic viscosity
496 BIOCHEMISTRY: SCHMITT ET AL. PROC. N. A. S.
dimensional lattice requisite for complement fixation is^ dependent on both the triple helix and the presence of telopeptides. If the triple helix is altered, for exam- ple, by collagenase or thermal treatment, the structure requisite for the three- dimensional aggregation is effected, and complement fixation is lost. The specificity of the TC: anti-TC reaction, however, resides in the telopeptides. The serological activity is diminished after protease treatment, the loss being greater after treatment with pronase which removes more peptide material than pepsin. In both cases, SLS aggregates of the digested TC appear normal under electron microscopic examination, and there is no physical evidence that the triple- helix body of the molecule has been altered. It is concluded that very small parts of the tropocollagen molecule in situations vulnerable to the action of proteases are the sites responsible for antigenicity. There is no conclusive evidence yet that the triple helix is continuous throughout the 2800 A length of the TC molecule; hence it might be supposed that the cross- links which are subject to protease attack could be situated in short regions of dis- order in the body of the helix. This possibility would appear (^) unlikely, however,
since pronase digests completely any denatured a-strand,'6 and one would expect
pronase to split the TC molecule at any site where the triple helix is interrupted. The remarkable stability of TC to pronase treatment seems a reflection of the in- tegrity of the triple-helix structure (^) throughout the 2800 A of its (^) length. On this as- sumption we suggest that the cross-links and the antigenic sites are (^) probably situ- ated external to the (^) triple-helix body. Since sonic irradiation or (^) protease treat- ment modifies the inherent linear (^) aggregative tendency of the (^) TC molecules, at least some^ of the^ telopeptide structures must be^ terminal in^ the macromolecule.
The tendency of TC to form a staggered array with overlap'2 may imply that other
structures, perhaps complementary to the terminal ones, are situated at points along the length of the helix; they may be attached to it by ester-type linkages.'8' ' It should be^ emphasized that TC solutions are not homogeneous; they contain molecules with both triple-a, ai-, and y-chains in the triple helix. No evidence yet shows that the antigenic property is^ common to all these TC variants. If it is feasible to differentiate immunologically between them, it may be possible to con-
firm or deny the postulated maturation sequence a to fi^ to 'y chains.
The hypothesis that in vivo control of collagen fibrogenesis is mediated by enzy- matic modification of telopeptides remains to be proved. However, the localization of antigenic activity in telopeptide structures provides a probe by which we antici- pate it will be possible to investigate collagen fibrogenesis, its homeostatic control, and (^) pathological defects in these control mechanisms. Summary.-Antibodies to purified calf-skin tropocollagen may be produced in rabbits. Complement fixation by antigen-antibody reaction is dependent upon the native structure of the tropocollagen molecules. The serological reaction can be reduced or abolished by protease attack which leaves the triple-helix body of the molecule undamaged. It is concluded that the antigenic response and the protease attack are directed against peptide appendages (^) external to the triple-helix body of the tropocollagen macromolecule. We (^) acknowledge with thanks the skillful technical assistance of Mr. J. W. Jacques, Miss Susan Bump, Miss Annelies Holzer, and Mrs. Elisabeth Myers. This (^) investigation was supported by grants AI-01469 and E-1940 from the National Institute of Allergy and Infectious Diseases, HE-
VOL. 51, 1964 BIOCHEMISTRY: RABIN AND (^) TROWN 497 08736 from the National Heart (^) Institute, and NB-0024 from the National Institute of Neuro- logical Diseases and Blindness, U.S. Public Health Service. Dr. M. P. Drake is supported by U.S. Public Health Service (^) fellowship 5F3AM-11,175 from the National Institute of Arthritis and Metabolic Diseases.
- (^) Publication no. 270 from the Graduate Department of Biochemistry, (^) Brandeis University. t Department of Biochemistry, Brandeis University. $ Rogosin^ Laboratories,^ Department^ of^ Medicine,^ Cornell^ University^ Medical^ Center. 1 Orekhovitch, V. N., A. A. (^) Tustanowski, K. D. (^) Orekhovitch, and N. (^) E. Plotnikova, Bio- khimiya, 13, 55 (1948). 2 Veis, A., J. (^) Anesey, and J. (^) Cohen, J. Am. (^) Leather Chemists' Assoc., 55, 548 (1960). (^3) Gross, J., J. H. (^) Highberger, and F. 0. (^) Schmitt, these PROCEEDINGS, 40, 679 (1954). 4 Boedtker, H., and P. (^) Doty, J. Am. (^) Chem. Soc., 78, 4267 (1956). (^5) Hall, C. B., and P. Doty, J. Am. Chem. Soc., 80, (^1269) (1958). (^6) Schmitt, F. (^) O., J. (^) Gross, and J. H. Highberger, Exptl. Cell Res., Suppl., 3, 329 (1955). (^7) Ramachandran, G. (^) N., in (^) Aspects of Protein Structure (New York: Academic Press, 1963), p. 39. 8 Piez, K. A., E. Weiss, and M. S. (^) Lewis, J. Biol. Chem., 235, 1987 (1960). (^9) Grassmann, W., K. (^) Hannig, and J. Engel, Z. Physiol. Chem., 324, 284 (1961). (^10) Altgelt, K., A. J. Hodge, (^) and F. 0. Schmitt, these PROCEEDINGS, 47, 1914 (1961). 1' Hodge, A. J., (^) and F. 0. Schmitt, these PROCEEDINGS, 44, 418 (1958). (^12) Hodge, A. J., (^) and J. A. (^) Petruska, in Aspects of Protein Structure (New York: Academic Press, 1963), p. (^) 289. (^13) Rubin, A. L., D. Pfahl, P. T. Speakman, P. F. Davison, and F. 0. Schmitt, Science, 139, 37 (1963). (^14) Hodge, A. J., J. H. Highberger, G. G. J. Deffner, and F. 0. Schmitt, these PROCEEDINGS, 46, 197 (1960). 16 Rubin, A.^ L., M. P. Drake, P. F. Davison, D. Pfahl, P. T. Speakman, and F. 0. Schmitt, in preparation. (^16) Drake, M. P., P. F. Davison, S. Bump, A. L. Rubin, and F. 0. Schmitt, in preparation. (^17) Wasserman, E., and L. Levine, J. Immunol., 87, 290 (1961). (^18) Gallop, P. M., S. Seifter, and E. Meilmain, Nature, 183, 1659 (1959). (^19) H6rmann, H., Leder, 11, 173 (1960). "0 (^) Orekhovitch, V. N., in Procollagens, Their Chemical (^) Structures, Properties, and Biological Role (Moscow, 1952).
INHIBITION OF CARBOXYDISMUTASE BY IODOACETAMIDE*
BY BRIAN R. RABINt AND PATRICK (^) W. TROWN BIO-ORGANIC CHEMISTRY GROUP, LAWRENCE RADIATION LABORATORY, UNIVERSITY OF (^) CALIFORNIA, BERKELEY Communicated by Melvin Calvin, January 9, 1964 The initial (^) step in the fixation of carbon (^) dioxide by autotrophic organisms is the reaction of carbon dioxide with RuDP to (^) give 3-phosphoglyceric acid." 2 This reaction is catalyzed by carboxydismutase, which is (^) inhibited by parachloromer- curibenzoate,3 suggesting that SH groups are (^) required for its (^) activity. In order
to define the function of these groups more precisely, we have investigated the
irreversible inhibition of the enzyme by iodoacetamide.
Experimental.-Enzyme: Crude carboxydismutase was prepared from isolated (^) spinach chloro- plasts as previously described.4 It was purified by fractional ammonium sulfate precipitation, followed by repeated gel filtration on Sephadex G-200.' The enzyme was stored as a precipitate
